The Role of the Retinoblastoma/E2F1 Tumor Suppressor Pathway in the
DNA Lesion Recognition Step of Nucleotide Excision Repair
Lisa A.
Y.
Julien
James M.
1
Stanford University School of Medicine , Stanford CA 94305
2
University of California at Davis, Internal Medicine , Sacramento CA 95817
Fig. 3. Global Genomic Repair Assay of Wildtype and
E2F1-/- MEFs following 10 J/m2 UV-C Irradiation
Fig. 7. A Putative E2F1 Binding Site in the Proximal
Promoter of Mouse XPC Gene
6-4PP Repair
+1
Percent Repair (%)
90
80
(-)
70
(+)
50
-484
-434
-384
-334
-284
-234
-184
-134
-84
-34
17
67
117
20
0
5
10
15
20
25
Time Post-Irradiation (Hrs)
CPD Repair
40
6-4PP Repair
90
Percent Repair (%)
35
2000
3000
4000
5000
6000
Exon 1
TCTACAGAGAAAGGGCGACGACGAGCGCGATGGGTAGACGGGCGGGTTGA
GAAACTCTGGGCCTACGGAGGTTTCAGGAGCAGGCCCGGCTACGGAAGGC
CGTGGTGACTCGGAAAAAAAGAAAAGGGCCATCCCACGGGAAGCGTGGAA
AAAGCGAGAAGCGAAAGCATTTCCTCTTCGAACGCCTCAGGTCAAACTTA
CCTGATCTACGTCGTCCGCCATGTTTCAAACGCTGCGCCCTTTCCACCTC
TCGCGGGAACAGGAACTCAGAAACCTTAGGCCGCCACGCACTGAAGGCGA
ACTATATTATTTTTGTCCCTTCGCGCCCTCGTAGTTTGCATGGGGGCGGG
GCTTCCTTCGAGGGCGTGGTCCTACCCTGGGCTGGGGGCGCGGCCAGGCG
TGGCCCGCCTCCGGGCGGGGCGGGCACGCGGAGACCTGCGGCGTCCTGCG
+1
CGGTAGTCCCGAGGAACGCCTCTGGCTAGCATGGCCCCAAAGCGCACCGC
AGACGGAAGGCGGCGGAAGCGGGGCCAGAAAACCGAGGACAACAAAGTAG
CCCGGCACGAGGAGAGCGTTGCGGGTGAGAGGCCGAGTCTGCAACATGCC
AGGCAGTGGTGGCTGAGGCTGGTGCGGGCGGCGGAGCGGATCTGCGCCTG
30
25
20
Fig. 8. E2F1 Chromatin Immunoprecipitation of XPC
Promoter following 10 J/m2 UV-C Irradiation
E2F1+/+
E2F1-/-
15
10
5
5.0
0
5
10
15
20
25
4.0
Time Post-Irradiation (Hrs)
Fig. 4. DDB2 mRNA Transcript Levels in Wildtype and
E2F1-/- MEFs following 10 J/m2 UV-C Irradiation
80
3.0
E2F1+/+
E2F1-/-
2.0
1.0
0.0
70
WT
Rb-/p107-/-;p130-/Rb-/-;p107-/-;p130-/-
60
50
40
DDB2/GAPDH (Fold over 0 Hr Time Pt.)
Percent Repair (%)
1000
(-166 to -155)
30
0
-1000
CTTCGCGCCCTC
E2F1+/+
E2F1-/-
40
-5
Fig. 1. Global Genomic Repair Assay of Wildtype and Rbdeficient MEFs following 10 J/m2 UV-C Irradiation
30
20
10
0
0
5
10
15
20
25
Time Post-Irradiation (Hrs)
0
3.0
2.5
30
WT
Rb-/p107-/-;p130-/Rb-/-;p107-/-;p130-/-
20
10
15
20
25
2.0
E2F1+/+
E2F1-/-
Conclusions
1.5
1.
2.
3.
4.
1.0
0
5
10
15
20
25
0.5
Fig. 5. A Putative E2F1 Binding Site in the Proximal
Promoter of Mouse DDB2 Gene (Nichols et al. 2003)
+1
(-)
10
Time Post-UVC Irradiation (Hrs)
Time Post-Irradiation (Hrs)
40
5
-1.0
CPD Repair
50
Percent Repair (%)
-2000
60
10
Results
-2000
-1000
1000
2000
3000
4000
5000
6000
Rb-deficient MEFs exhibit increased global genomic repair.
Rb-deficient MEFs express higher basal level of DDB2.
E2F1-deficient MEFs exhibit decreased global genomic repair.
E2F1-deficient MEFs exhibit decreased DNA damage-inducible
expression of DDB2 and XPC following UV-C irradiation.
5. Proximal promoter region of the mouse XPC gene contains a putative E2F
binding site.
6. E2F1 binds XPC promoter in the native chromatin structure.
7. The Rb/E2F1 tumor suppressor pathway plays a regulatory role in the
DNA lesion recognition step of nucleotide excision repair.
(+)
0
0
5
10
15
20
25
Exon 1
TTTGGCGC
-10
Exons 2 & 3
References
Exons 2 & 3
(+35 to +42)
Time Post-Irradiation (Hrs)
Fig. 2. DDB2 and PCNA mRNA Transcript Levels in
Wildtype and Rb-deficient MEFs
Fig. 6. XPC mRNA Transcript Levels in Wildtype and
E2F1-/- MEFs following 10 J/m2 UV-C Irradiation
5
9
8
7
6
DDB2/GAPDH
PCNA/GAPDH
5
4
3
2
1
0
WT
Rb-/-
Rb-/;p107-/-
MEFs
p107-/;p130-/-
TKO
XPC/GAPDH (Fold over 0 Hr Time Pt.)
Fold Activation (over WT)
Cell Lines
Primary Rb-/-, Rb-/-;p107-/-, p107-/-;p130-/-, and Rb-/-;p107-/-;p130-/MEFs were derived from chimeric embryos (Sage et al. 2000). Wildtype
(WT) and E2F1-/- MEFs (Field et al. 1996) were generously provided by Drs.
Rosalie Sears and Charles Lopez (Oregon Health and Science University).
Results (cont'd)
0
10
Materials and Methods
1
Ford
Global Genomic Repair
Repair of CPDs and 6-4PPs was measured using an enzyme-linked
immunosorbent assay (ELISA). Briefly, exponentially growing cells were
irradiated with 10 J/m2 UV-C, the genomic DNA was isolated and distributed
in triplicate onto microtiter plates precoated with 0.003% protamine sulfate.
DNA lesions were detected with either 1:5000 TDM-2 (for CPDs) or 1:5000
64M-2 (for 6-4PPs) (Mori et al. 1991). The signals were amplified and
subsequently developed with 3,5,3',5'-tetramethylbenzidine (TMB). The
reactions were quantified at 450nm on a microplate reader.
Quantitative RT-PCR
Real Time RT-PCR was used to evaluate basal and inducible NER transcripts
in MEFs via an ABI PRISM 7900 Sequence Detection System (Applied
Biosystems).
Promoter Analysis for Putative E2F Binding Sites
Putative E2F1 binding sites in the mouse XPC gene were determined using a
computer
algorithm
and
weight-substitution
matrix:
http://compel.bionet.nsc.ru/FunSite/SiteScan.html (Kel et al. 2001).
Chromatin Immunoprecipitation (ChIP) Assay
ChIP assays were performed using the Chromatin Immunoprecipitation Assay
Kit (Upstate) according to manufacturer's protocol. Mouse DNA was
immunoprecipitated with C-20 E2F1 antibody (Santa Cruz Biotechnology).
Enriched DNA was purified and PCR-amplified via ABI PRISM 7900. All
signals were normalized to levels found 5000 base pairs upstream of the
Background
Recently, Berton and colleagues established a function of E2F1 in promoting
DNA repair (Berton et al. 2005). Mice containing homozygous knockout of
E2F1 exhibited enhanced keratinocyte apoptosis following UV-B irradiation
whereas mice with transgenic overexpression of E2F1 displayed decreased
epidermal apoptosis. Furthermore, E2F1-/- mice were deficient for the
removal of DNA photoproducts while transgenic mice exhibited an enhanced
level of repair. Accordingly, the suppression of apoptosis by E2F1 is related
to an increase in DNA repair. While the study revealed a putative role of
E2F1 in the repair of UV-induced damaged DNA, the mechanism by which
E2F1 stimulates NER remained unclear. One possibility is that E2F1
transcriptionally regulates one or more rate-limiting factors involved in the
DNA lesion recognition step of NER. Recently, Prost and colleagues
demonstrated that E2F1 is a transcriptional regulator for DDB2 (Prost et al.
2006). Here, we established that E2F1 likewise is a transcriptional regulator
of XPC, and plays a fundamental role in the DNA lesion recognition step of
murine NER.
1
Sage ,
Results (cont'd)
100
A number of DNA microarrays and computer-assisted promoter analyses
strongly suggest that E2F may be involved in several DNA repair pathways,
including nucleotide excision repair (NER). Among the eight members of the
E2F family (E2F1-E2F8), only E2F1 is phosphorylated and stabilized upon
DNA damage. Furthermore, protein-protein interactions between E2F1 and a
number of repair machineries have been established. Although the functional
significance of these independent findings is not well understood, the
collective body of evidence strongly implicates a role of E2F1 in DNA
damage repair.
1
Chen ,
Materials and Methods (cont'd)
Abstract
The retinoblastoma (Rb)/E2F1 tumor suppressor pathway plays a major role
in the regulation of mammalian cell cycle progression and cell proliferation.
The Rb protein, along with closely related proteins p107 and p130, exerts its
anti-proliferative effects by binding to the E2F family of transcription factors
known to regulate essential genes throughout the cell cycle. The disruption of
the Rb/E2F pathway is regarded as a frequent, if not universal, feature of
tumorigenesis. Recent lines of evidence suggest that the Rb/E2F1 pathway
may be involved in DNA damage repair. To determine if deregulation of the
Rb/E2F1 pathway can affect nucleotide excision repair (NER), we assayed
the ability of wildtype (WT) and various Rb-deficient mouse embryonic
fibroblasts (MEFs) to repair DNA lesions following UV-C irradiation.
Rb-/-;p107-/-;p130-/- triple knockout (TKO) MEFs repaired both cyclobutane
pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts
(6-4PPs) at higher efficiency than did WT MEFs, while Rb-/- single mutant
MEFs and p107-/-;p130-/- double mutant MEFs exhibited intermediate repair
phenotypes. The expression of damaged DNA binding gene 2 (DDB2)
involved in the DNA lesion recognition step of NER was elevated in
Rb-deficient MEFs. To determine if the enhanced NER in the absence of the
Rb gene family is due to the derepression of E2F1, we assayed the ability of
E2F1-deficient cells to repair UV-induced DNA lesions and demonstrated that
E2F1-/- MEFs are impaired for the removal of CPDs and 6-4PPs.
Furthermore, WT MEFs induced a higher expression of DDB2 and xeroderma
pigmentosum group C (XPC) transcript levels than did E2F1-/- MEFs at 16
and 24 hours following UV-C irradiation. This data is consistent with the
recent finding that the DDB2 gene, and as we currently demonstrate, the XPC
gene, contain putative E2F binding sites in their promoters. Chromatin
immunoprecipitation (ChIP) assay confirms E2F1 binding to the XPC
promoter in the native chromatin structure. Our study here suggests a
regulatory role of the Rb/E2F1 tumor suppressor pathway in the DNA lesion
recognition step of nucleotide excision repair.
1
McPherson , Aubrey
ChIP Binding (Arbitrary Units)
Patrick S.
1,2
Lin ,
4
1.
2.
3.
4.
5.
6.
7.
Berton, T.R. et al. (2005). Oncogene 24, 2449-2460.
Prost, S. et al. (2006). Oncogene (epub ahead of print).
Sage, J. et al. (2000). Genes Dev. 14, 3037-3050.
Field, S.J. et al. (1996). Cell 85, 549-561.
Mori, T. et al. (1991). Photochem. Photobiol. 54, 225-232.
Kel, A.E. et al. (2001). J. Mol. Biol. 309, 99-120.
Nichols, A.F. et al. (2003). Nucl. Acids Res. 31, 562-569.
Acknowledgments
3
E2F1+/+
E2F1-/2
1
0
5
10
15
0
Time Post-Irradiation (Hrs)
20
25
We thank members of the Ford Lab for many fruitful discussions and insights.
This work was supported by National Institutes of Health-National Research
Service Award PHS NRSA 5T32 CA09302-27 (to P.S.L), American Cancer
Society Postdoctoral Fellowship Award PF-06-037-01-GMC (to P.S.L.), and
National Institutes of Health Award RO1 CA108794 (to J.M.F.).
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